The tight binding of a spermatozoan to the zona pellucida surrounding an egg is a critical event in fertilization. Thus, the identification of the molecules responsible for this binding is of fundamental importance. It is generally agreed that in the mouse, receptors on the sperm plasma membrane bind to the nonreducing termini of specific O-linked glycans on zona pellucida glycoprotein-3 (ZP3). Current debate centers on the identity of the receptor(s) on sperm and the structure(s) of the glycans they bind. To gain insight into the structures of the ligands bound by sperm, we analyzed the activities of a series of small glycans of known structure in a competitive sperm-zona binding assay. Results showed that a beta- Gal-capped but not a beta-C1cNAc-capped trisaccharide was a low activity competitive inhibitor (ED50=42muM). Addition of a nonreducing terminal alpha3-Gal to the beta-Gal-capped glycan increased activity 8-fold. However, additional of an alpha3-fucose residue to either the beta-Gal- or the alpha-Gal-capped trisaccharide produced a highly effective competitive inhibitor (ED50 approximately 0.45muM for both glycans). Lastly, mixing experiments with pairs of competitive inhibitors demonstrated additive effects when saturating does of fucosylated, Gal-capped glycans and a beta-Gal-capped glycan were added to sperm and eggs. These results lead to the hypotheses that fucosylated, Gal-capped glycans have high affinity for a receptor on sperm, that beta-Gal-capped glycans have a lower affinity for a receptor and that these two classes of glycans bind different receptors on the sperm surface. To test these hypotheses, we propose to synthesize and radiolabel two neoglycoproteins, and use these probes to directly determine whether they bind separate receptors. We will also examine how changes in a glycan's structure affects its affinity for these receptors. We next propose to test the function of these receptors by examining whether their expression is affected by capacitation, whether they mediate the acrosome reaction or are required for fertilization. We will then identify the glycan receptors on the sperm surface by cross linking the neoglcoproteins to their respective receptors and examining the cross-linked protein. Finally, using antibodies, glycosyltransferases, glycosidases and lectins with defined specificities for glycan structures, we propose to determine whether glycans with specific fucosylated, Gal-capped and/or beta-Gal-capped termini are present on ZP3. Completion of these experiments will provide substantial insight into the structures and functions of the molecules on murine sperm and the zona pellucida that mediate critical events in fertilization.